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Human papilloma virus 18 L1 (HPV18L1) polynucleotide sequence and its expression vector, host cell and use

A polynucleotide and host cell technology, applied in the field of HPV18L1 polynucleotide sequence, can solve the problems of general public acceptance

Active Publication Date: 2012-10-10
天津昕因达生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The human papillomavirus vaccines developed by Merck using brewer's yeast and GlaxoSmithKline using insect cell-baculovirus are both on the market, mainly for immunization in Europe. High, not acceptable to the general public

Method used

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  • Human papilloma virus 18 L1 (HPV18L1) polynucleotide sequence and its expression vector, host cell and use
  • Human papilloma virus 18 L1 (HPV18L1) polynucleotide sequence and its expression vector, host cell and use
  • Human papilloma virus 18 L1 (HPV18L1) polynucleotide sequence and its expression vector, host cell and use

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Experimental program
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Effect test

Embodiment 1

[0053] Embodiment 1: HPV18L1 gene sequence optimization

[0054] Convert the full-length 508 amino acids of the HPV18L1 amino acid sequence into the nucleotide sequence of Hansenula's preferred codon pattern. Among the preferred demerging codons, Hansenula's preference is the first codon, and Hansen's preferred codons are mainly used. Yeast prefers the first and second codons, and in a few cases, the third codon is used to complete local adjustments. DNAMAN software is used to avoid excessively long complementary sequences and repetitive sequences, and RNA structure prediction is used to avoid excessively long and complex hairpin structures. , to exclude strong secondary structures and increase the internal stability of specific molecules. The nucleotide sequence of the gene also avoids sequences that may adversely affect gene transcription and translation efficiency, such as intron splicing sequences, transcription termination sequences, internal promoter sequences (TATA) and...

Embodiment 2

[0056] Embodiment 2: HPV18L1 gene and HPV18L1 mutant gene expression vector construction

[0057] The DNA sequence shown in Sequence 2 in the Sequence Listing was synthesized by a fully artificial method, and recognition sites for restriction endonucleases EcoR I and BamH I were added to both ends of the sequence. The synthesized DNA sequence was digested with EcoR I and BamH I, and the digested fragment was recovered by electrophoresis, and stored at -20°C for future use.

[0058] Using the genomic DNA of Hansenula polymorpha CGMCC 2.2497 as a template, clone the 1.5kb methanol oxidase gene (MOX) promoter, the 350bp methanol oxidase gene (MOX) terminator, and the 1.0kb self-replicating sequence HARS; and from YIp5 (GeneBank Accession NO.L09157) plasmid cloned the 1.1kb Saccharomyces cerevisiae uracil gene ScURA3; after connecting the above four parts, it was inserted into the multiple cloning site of the pBluescrip II plasmid to construct the shuttle vector pMPT-02.

[0059]...

Embodiment 3

[0062] Example 3: Electrotransformation of Hansenula Competent Host Cells with Expression Vectors

[0063] The vector pMPT-HPV18L1 was digested with Sac I to linearize the vector. The linearized vector was transformed into Hansenula polymorpha (Hansenula polymorpha) CGMCC NO.1218 by electroporation method (LN-101 gene pulse introduction instrument produced by Tianjin University of Technology, 1.5kV, 50μF, 200Ω, 3-5mSec). Transformants were selected on a medium plate containing 1.34g / 100ml YNB (yeast nitrogen base medium), 2g / 100ml glucose.

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Abstract

The invention relates to a human papilloma virus 18 L1 (HPV18L1) polynucleotide sequence and its expression vector, host cell and use and especially relates to an amino acid sequence of an HPV L1 capsid protein, a synthetic nucleotide sequence for coding the amino acid sequence, a recombinant expression vector containing the synthetic nucleotide sequence, and a hansenula polymorpha expression host strain containing the synthetic nucleotide sequence. The invention also relates to a use of an HPV18L1 protein composed of the amino acid sequence and derivatives of the amino acid sequence in preparation of vaccines. Through modification of a nucleotide sequence of a gene of an HPV18L1 wide-type virus, a recombinant HPV18L1 capsid protein can be highly expressed in a hansenula polymorpha expression system and thus hansenula polymorpha expression system-based industrial production of an HPV18L1 capsid protein is realized. Compared with the existing eukaryotic expression systems, the HPV18L1 polynucleotide sequence and its expression vector and host cell have advantages of higher yield and lower cost.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to an HPV18L1 polynucleotide sequence, and also includes an expression vector, a host cell and the application of the polynucleotide sequence, the expression vector, and the host cell in pharmaceuticals and preparations. Background technique [0002] Infection with papillomaviruses has been found in a variety of species including sheep, dogs, rabbits, monkeys, cattle and humans. Human papillomavirus (HPV for short) has been separated into hundreds of types. If the identity of the L1 gene and the L1 sequence of the previously identified type is less than 90%, then this type can be determined as a new type. A subtype is defined if the L1 amino acid sequence identity is between 90 and 98%, and a variant [compared to the prototype (parental type)] if the identity is above 98%. [0003] Human papillomavirus is a kind of DNA virus with strict host range and tissue specifici...

Claims

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Application Information

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IPC IPC(8): C12N15/37C07K14/025C12N15/81C12N1/19A61K39/12A61P31/20C12R1/78
Inventor 王昌华杨珺齐怀丰李建王艳侠汪志良张涛宋立娜王玉香孙长海汪和睦
Owner 天津昕因达生物技术有限公司
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